It is common to preserve blood, body tissue, and semen by freezing. Example of methods and devices used for the freezing and storing of blood and body tissue are illustrated in U.S. Pat. No. 4,630,448, Bilstad, et al., U.S. Pat. No. 3,952,536, Faust, et al., U.S. Pat. No. 4,251,995, Pert, et al., U.S. Pat. No. 3,431,172, Rajamannam, and U.S. Pat. No. 4,865,871, Livesey, et al.
However, the freezing of spermatozoa involves entirely different problems than those faced in connection with the freezing of blood or tissue. Blood and tissue are not as easily damaged during the freezing or subsequent thawing process as are spermatozoa.
The three functions that are particularly critical and specific to spermatozoa are (1) the innate ability of spermatozoa to locate the ovum, (2) the self mobilization of the spermatozoa, and (3) the ability of the spermatozoa to attach to the ovum and enter through barriers to fertilize the ovum.
The ability of the spermatozoa to move from the cervix to the ovum located high in the ovaduct is a very specialized function that is easily disrupted by changes in temperature. Other cells that are preserved by freezing (i.e., blood cells), depend on cardiovascular circulation to transport them where they are needed.
The spermatozoa has a tail that rotates so that the cell is moved in a straightforward direction. When a spermatozoa is cooled too rapidly without proper preparation and the use of extenders, the motility apparatus in the tail may be damaged to change the motility of the spermatozoa. Even a slight change in the motility can result in the spermatozoa moving in circles or to inhibit its motility completely. In either case the spermatozoa are incapable of reaching the ovum and achieving fertilization.
The spermatozoa has the ability to enter the ovum after crossing three barriers (cumulus cells, zona pellucida, and the ovum's cell membrane). To accomplish this task it requires the motility generated by the tail and special enzymes located at the head of the spermatozoa under a membrane referred to as the acrosomal cap. The acrosomal cap is dissolved away shortly before the spermatozoa reaches the ovum. The process is referred to as capacitation. If the acrosomal cap is damaged during the freezing process, then the enzymes may be released too soon, or the cap may be unable to undergo capacitation. There has also been speculation that under the acrosomal cap there may be specialized receptors that enable the spermatozoa to attach to the ovum's cell membrane and allow entrance into the ovum. This is referred to as fertilization. How the cooling, freezing, and thawing techniques affect this final step is still unknown.
Examples in the prior art of patents relating to the process for the preservation of semen are set forth in U.S. Pat. No. 3,973,003, Colas, U.S. Pat. No. 3,816,249, Bhattacharya, U.S. Pat. No. 3,791,384, Richter, et al., and U.S. Pat. No. 3,940,943, Sikes, et al.
Currently spermatozoa for use in the insemination of such animals as horses and cattle are preserved frozen in straws that are less than 4 mm in diameter. In preparing semen for freezing it is common practice to add a diluent, then centrifuge the mix to remove seminal plasma. Thereafter the semen is mixed with cryoprotectant or an extender that protects the semen to some extent during the freezing and thawing process.
The disadvantage of the use of straws to preserve frozen spermatozoa, which is the current state of the art, is that the spermatozoa must be either packaged so concentrated so as not to allow enough extender to be incorporated to properly protect the spermatozoa during freezing, or multiple straws must be used to inseminate the female. As a practical matter, this technique is only successful in species where a low number of spermatozoa are sufficient to provide fertility such as in cattle.
Another disadvantage of the use of straws is the temperature to thaw the semen to retain fertility is relatively high, even though the time required to thaw the sample may be short (75.degree. C. for 7.0 seconds). It has long been established that temperatures above body temperature (37.degree. C.) decrease the longevity of spermatozoa. Faulkner, et al. Artificial Insemination in Veterinarian Endocrinology and Reproduction, edited by L. E. McDonald, 2nd edition 1977, described the thermal limit of spermatozoon's viability from 50.degree. C. to temperatures below -196.degree. C. (with cryoprotectants). Therefore, the outer layers of spermatozoa may undergo thermal damage and be unable to achieve fertility. After the spermatozoa are thawed with the high temperature, the sample must be rushed to the female for insemination to reduce the recooling of the spermatozoa as this may result in damage to the spermatozoa.